The conjugation of water-soluble polyalkylene oxide (“PAO”) with therapeutic moieties such as proteins and polypeptides is known. See, for example, U.S. Pat. No. 4,179,337, the disclosure of which is hereby incorporated by reference. The '337 patent discloses that physiologically active polypeptides modified with PEG circulate for extended periods in vivo, have reduced immunogenicity and antigenicity.
To conjugate PAO with other compounds, the hydroxyl end-groups of the polymer must first be converted into reactive functional groups. This process is frequently referred to as “activation” and the product is called an activated polyalkylene oxide or activated PAO.
For the most part, research has been directed to covalent attachment of PAO's to epsilon amino groups of proteins, enzymes and polypeptides. Covalent attachment of PAO's to lysine amino groups has been effected by linking groups such as succinoyl-N-hydroxysuccinimide ester, as disclosed by Abuchowski et al., Cancer Biochem. Biophys., 7, 175-86 (1984), azlactones, aryl imidates and cyclic imide thiones. See U.S. Pat. Nos. 5,298,643, 5,321,095, and 5,349,001, for example. The contents of each of the foregoing patents are hereby incorporated by reference herein. PAO's have also been activated with hydrazine groups in order to couple the polymer to activated carbohydrate groups.
In addition, the conversion of terminal hydroxy groups of PAO's, such as polyethylene glycol (“PEG”), to carboxylic acids has also been reported. PEG-acids are useful in at least two regards. First, carboxylic acid derivatives can be  used directly to conjugate nucleophiles via available hydroxyl or amino moieties. Secondly, PEG carboxylic acids can be used as intermediates to form other types of activated polymers. For example, mPEG carboxylic acids can be converted to the succinimidyl ester derivative via N-hydroxysuccinimide and a condensing agent such as diisopropyl carbodiimide. Other activated PAO's can be prepared by reaction of the active ester with hydrazine to produce PAO-hydrazide derivatives.
Co-owned U.S. Pat. No. 5,605,976 (the '976 patent), incorporated by reference herein, solved many previous difficulties in preparing polyalkylene oxide carboxylic acids. The '976 patent taught a process for preparing PAO carboxylic acids by reacting a PAO (i.e., PAO-OH) with a tertiary alkyl haloacetate in the presence of a base to form a tertiary alkyl ester of PAO, and then reacting the PAO tertiary alkyl ester with an acid, to form the desired polyalkylene oxide carboxylic acid.
In the time since the methods of the '976 patent were developed, a need for further improvements arose. For example, with improvements in NMR instrumentation, it became apparent that batches of PEG-acid still contained ˜5% PEG-OH impurity. In addition, it was determined that levels of contamination with the native PEG-OH tended to increase with the molecular weight of the polymer, and with the use of disubstituted and branched PEG polymers. In addition, the processes taught by the '976 patent required at least 18 hours of reaction time, as well as refluxing and rotary evaporation of the reaction solvent.
For at least the foregoing reasons, there remains a longstanding need in the art for more rapid, and therefore more economical, methods for preparing PAO carboxylic acids, as well a need for methods for producing PAO acids and intermediates of much higher purity that are free of any detectable PAO-OH contamination. The present invention addresses these needs.